Juvenile Myelomonocytic Leukemia (JMML) is a lethal myeloproliferative disorder (MPN) in children that is characterized by the growth hypersensitivity and RAS pathway hyperactivity of myeloid cells in response to the cytokine granulocyte-macrophage colony stimulating factor (GM-CSF). JMML resists treatment by chemotherapy and frequently relapses following hematopoietic stem cell (HSC) transplantation. Therefore new therapies are crucially needed to treat this disorder. JMML frequently presents in infants with non-specific bone marrow findings and significant extramedullary involvement, in the spleen, liver, skin, brain, and other tissues. These findings suggest that tissue-specific hematopoietic niches may play a role in this development of this disorder. Our lab and others have recently shown that embryonic hematopoiesis originates in the yolk sac from a developmental program that is distinct from and independent of adult HSCs. Yolk sac HSC-independent myeloid progenitors are now know to persist after birth and throughout adulthood as tissue-resident macrophages. These macrophage populations maintain themselves via local proliferation independently of bone marrow contributions and resist radiation. These findings lead up to hypothesize that HSC-independent yolk sac progenitors contribute to the development of JMML. Our preliminary data for this project shows that yolk sac progenitors have features of MPN in a mouse model of JMML. We propose to extend our findings and determine if these MPN features persist in the descendants of these yolk sac progenitors - the tissue resident macrophages of the brain, liver, and epidermis. We will isolate macrophage populations from each of these tissues and measure their proliferative potential and RAS pathway activity following stimulation with GM-CSF. We will equally confirm whether the phenotype of these mutant tissue macrophages-which resist contributions from bone marrow precursors-will change following bone marrow transplantation. Finally, we will test the hypothesis whether mutations that are restricted to the HSC-independent yolk sac myeloid lineages are sufficient to produce MPN. This will be the first study to consider the embryonic origin of JMML and the contribution of HSC-independent hematopoiesis to post-natal disease.